Doc.: INS SAG1ULTRA.CE Page 1 of 9 Rev.

: 0 Date: 10/2008

HBsAgone
Version ULTRA
Fourth generation Enzyme
Immunoassay (ELISA)
for the determination of
Hepatitis B surface Antigen or HBsAg
in human serum and plasma
- for “in vitro” diagnostic use only -

DIA.PRO
Diagnostic Bioprobes Srl
Via Columella n° 31
20128 Milano - Italy
Phone +39 02 27007161
Fax +39 02 26007726
e-mail: [email protected]

REF SAG1ULTRA.CE
96/192/480/960 Tests
 Doc.: INS SAG1ULTRA.CE Page 2 of 9 Rev.: 0 Date: 10/2008

Hepatitis B surface Antigen or HBsAg is the most important

HBsAg One version ULTRA protein of the envelope of Hepatitis B Virus, responsible for
acute and chronic viral hepatitis.
The surface antigen contains the determinant “a”, common to all
A. INTENDED USE the known viral subtypes, immunologically distinguished by two
Fourth generation Enzyme Immunoassay (ELISA) for the distinct subgroups (ay and ad).
one-step determination of Hepatitis B surface Antigen or The ability to detect HBsAg with high sensitive immunoassays in
HBsAg in human plasma and sera. the last years has led to an understanding of its distribution and
The kit may be used for the screening of blood units, is able to epidemiology worldwide and to radically decrease the risk of
detect HBsAg mutants and find application in the follow-up of infection in transfusion.
HBV-infected patients.
For “in vitro” diagnostic use only.
C. PRINCIPLE OF THE TEST
A mix of mouse monoclonal antibodies specific to the
B. INTRODUCTION determinants “a”, “d” and “y” of HBsAg is fixed to the surface of
microwells. Patient’s serum/plasma is added to the microwell
The World Health Organization (WHO) defines Hepatitis B Virus
together with a second mix of mouse monoclonal antibodies,
infection as follows:
conjugated with Horseradish Peroxidase (HRP) and directed
against a different epitope of the determinant “a” and against
“Hepatitis B is one of the major diseases of mankind and is a serious
global public health problem. Hepatitis means inflammation of the liver, “preS”.
and the most common cause is infection with one of 5 viruses, called The specific immunocomplex, formed in the presence of HBsAg
hepatitis A,B,C,D, and E. All of these viruses can cause an acute disease in the sample, is captured by the solid phase.
with symptoms lasting several weeks including yellowing of the skin and At the end of the one-step incubation, microwells are washed to
eyes (jaundice); dark urine; extreme fatigue; nausea; vomiting and remove unbound serum proteins and HRP conjugate.
abdominal pain. It can take several months to a year to feel fit again. The chromogen/substrate is then added and, in the presence of
Hepatitis B virus can cause chronic infection in which the patient never
captured HBsAg immunocomplex, the colorless substrate is
gets rid of the virus and many years later develops cirrhosis of the liver or
liver cancer. hydrolyzed by the bound HRP conjugate to a colored end-
product. After blocking the enzymatic reaction, its optical density
HBV is the most serious type of viral hepatitis and the only type causing is measured by an ELISA reader.
chronic hepatitis for which a vaccine is available. Hepatitis B virus is The color intensity is proportional to the amount of HBsAg
transmitted by contact with blood or body fluids of an infected person in present in the sample.
the same way as human immunodeficiency virus (HIV), the virus that The version ULTRA is particularly suitable for automated
causes AIDS. However, HBV is 50 to 100 times more infectious than HIV. screenings and is able to detect “s” mutants.
The main ways of getting infected with HBV are: (a) perinatal (from
mother to baby at the birth); (b) child- to-child transmission; (c) unsafe
injections and transfusions; (d) sexual contact.

Worldwide, most infections occur from infected mother to child, from child D. COMPONENTS
to child contact in household settings, and from reuse of un-sterilized The standard configuration contains reagents to perform 192
needles and syringes. In many developing countries, almost all children tests and is made of the following components:
become infected with the virus. In many industrialized countries (e.g.
Western Europe and North America), the pattern of transmission is
1. Microplate MICROPLATE
different. In these countries, mother-to-infant and child-to-child
transmission accounted for up to one third of chronic infections before n° 2. 12 strips of 8 breakable wells coated with anti HBsAg,
childhood hepatitis B vaccination programmes were implemented. affinity purified mouse monoclonal antibodies, specific to “a”, “y”
However, the majority of infections in these countries are acquired during and “d” determinants, and sealed into a bag with desiccant.
young adulthood by sexual activity, and injecting drug use. In addition,
hepatitis B virus is the major infectious occupational hazard of health 2. Negative Control CONTROL -
workers, and most health care workers have received hepatitis B vaccine.
1x4.0ml/vial. Ready to use control. It contains goat serum, 10
mM phosphate buffer pH 7.4+/-0.1, 0.09% Na-azide and 0.1%
Hepatitis B virus is not spread by contaminated food or water, and cannot
be spread casually in the workplace. High rates of chronic HBV infection
Kathon GC as preservatives. The negative control is pale yellow
are also found in the southern parts of Eastern and Central Europe. In the color coded.
Middle East and Indian sub-continent, about 5% are chronically infected.
Infection is less common in Western Europe and North America, where 3. Positive Control CONTROL +
less than 1% are chronically infected. 1x4.0ml/vial. Ready to use control. It contains goat serum,
non infectious recombinant HBsAg, 10 mM phosphate buffer pH
Young children who become infected with HBV are the most likely to 7.4+/-0.1, 0.02% gentamicine sulphate and 0.1% Kathon GC as
develop chronic infection. About 90% of infants infected during the first preservatives. The positive control is color coded green.
year of life and 30% to 50% of children infected between 1 to 4 years of
age develop chronic infection. The risk of death from HBV-related liver
cancer or cirrhosis is approximately 25% for persons who become 4. Calibrator CAL ….
chronically infected during childhood. Chronic hepatitis B in some n° 2 vials. Lyophilized calibrator. To be dissolve d with EIA
patients is treated with drugs called interferon or lamivudine, which can grade water as reported in the label. Contains fetal bovine
help some patients. Patients with cirrhosis are sometimes given liver nd
serum, non infectious recombinant HBsAg at 0.5 IU/ml (2
transplants, with varying success. It is preferable to prevent this disease WHO international standard for HBsAg, NIBSC code 00/588),
with vaccine than to try and cure it.
10 mM phosphate buffer pH 7.4+/-0.1, 0.02% gentamicine
Hepatitis B vaccine has an outstanding record of safety and sulphate and 0.1% Kathon GC as preservatives.
effectiveness. Since 1982, over one billion doses of hepatitis B vaccine Note: The volume necessary to dissolve the content of the
have been used worldwide. The vaccine is given as a series of three vial may vary from lot to lot. Please use the right volume
intramuscular doses. Studies have shown that the vaccine is 95%
reported on the label .
effective in preventing children and adults from developing chronic
infection if they have not yet been infected. In many countries where 8%
to 15% of children used to become chronically infected with HBV, the rate 5. Wash buffer concentrate WASHBUF 20X
of chronic infection has been reduced to less than 1% in immunized 2x60ml/bottle. 20X concentrated solution. Once diluted,
groups of children. Since 1991, WHO has called for all countries to add the wash solution contains 10 mM phosphate buffer pH 7.0+/-
hepatitis B vaccine into their national immunization programs.” 0.2, 0.05% Tween 20 and 0.1% Kathon GC.
 Doc.: INS SAG1ULTRA.CE Page 3 of 9 Rev.: 0 Date: 10/2008

6. Enzyme Conjugate Diluent CONJ DIL F. WARNINGS AND PRECAUTIONS

2x16ml/cassette. Ready to use and red color coded regent. 1. The kit has to be used by skilled and properly trained
It contains 10 mM Tris buffer pH 6.8+/-0.1, 1% normal mouse technical personnel only, under the supervision of a medical
serum, 5% BSA, 0.1% Kathon GC and 0.02% gentamicine doctor responsible of the laboratory.
sulphate as preservatives. The solution is normally opalescent. 2. When the kit is used for the screening of blood units and
blood components, it has to be used in a laboratory certified and
7. Enzyme Conjugate CONJ 20X qualified by the national authority in that field (Ministry of Health
2x1ml/vial. 20X concentrated reagent. It contains or similar entity) to carry out this type of analysis.
Horseradish Peroxidase (HRP) labeled mouse monoclonal 3. All the personnel involved in performing the assay have to
antibodies to HBsAg, determinant “a” and “preS”, 10 mM Tris wear protective laboratory clothes, talc-free gloves and glasses.
buffer pH 6.8+/-0.1, 5% BSA, 0.1% Kathon GC and 0.02% The use of any sharp (needles) or cutting (blades) devices
gentamicine sulphate as preservatives. should be avoided. All the personnel involved should be trained
in biosafety procedures, as recommended by the Center for
8. Chromogen/Substrate SUBS TMB Disease Control, Atlanta, U.S. and reported in the National
2x25ml/cassette. It contains a 50 mM citrate-phosphate Institute of Health’s publication: “Biosafety in Microbiological and
buffered solution at pH 3.5-3.8, 4% dimethylsulphoxide, 0.03% Biomedical Laboratories”, ed. 1984.
tetra-methyl-benzidine (TMB) and 0.02% hydrogen peroxide 4. All the personnel involved in sample handling should be
(H2O2). vaccinated for HBV and HAV, for which vaccines are available,
Note: To be stored protected from light as sensitive to safe and effective.
strong illumination. 5. The laboratory environment should be controlled so as to
avoid contaminants such as dust or air-born microbial agents,
9. Sulphuric Acid H2SO4 O.3 M when opening kit vials and microplates and when performing the
1x25ml/cassette. It contains 0.3 M H2SO4 solution. test. Protect the Chromogen (TMB) from strong light and avoid
vibration of the bench surface where the test is undertaken.
Note: Attention: Irritant (Xi R36/38; S2/26/30)
6. Upon receipt, store the kit at 2..8°C into a tem perature
10. Container caps: n° 5 controlled refrigerator or cold room.
7. Do not interchange components between different lots of
the kits. It is recommended that components between two kits
11. Package insert
of the same lot should not be interchanged.
12. Plate sealing foils: n° 4 8. Check that the reagents are clear and do not contain
visible heavy particles or aggregates. If not, advise the
laboratory supervisor to initiate the necessary procedures for kit
replacement.
9. Avoid cross-contamination between serum/plasma
Important note: samples by using disposable tips and changing them after each
Only upon specific request , Dia.Pro can supply reagents for 96, sample.
480, 960 tests , as reported below: 10. Avoid cross-contamination between kit reagents by using
disposable tips and changing them between the use of each
Microplates N°1 N°5 N°10 one.
Negative Control 1x2ml/vial 1x10ml/vial 1x20ml/vial 11. Do not use the kit after the expiration date stated on the
Positive Control 1x2ml/vial 1x10ml/vial 1x20ml/vial external container and internal (vials) labels. A study conducted
Calibrator N° 1 vial N° 5 vials N° 10 vials
Wash buffer concentrate 1x60ml/vial 2x150ml/vial 4x150ml/vial on an opened kit has not pointed out any relevant loss of activity
Enzyme conjugate 1x0.8ml/vial 1x4ml/vial 2x4ml/vial up to 6 re-use of the device and up to 6 months.
Conjugate Diluent 1x16ml/vial 1x80ml/vial 2x80ml/vial 12. Treat all specimens as potentially infective. All human
Chromogen/Substrate 1x25ml/vial 1x125ml/vial 2x125ml/vial serum specimens should be handled at Biosafety Level 2, as
Sulphuric Acid 1x15ml/vial 1x80ml/vial 2x80ml/vial recommended by the Center for Disease Control, Atlanta, U.S.
Plate sealing foils N° 2 N° 10 N° 20 in compliance with what reported in the Institutes of Health’s
Package insert N° 1 N° 1 N° 1 publication: “Biosafety in Microbiological and Biomedical
Number of tests 96 480 960
Laboratories”, ed. 1984.
Code SAG1ULTRA.CE .96 .480 .960
13. The use of disposable plastic-ware is recommended in the
preparation of the liquid components or in transferring
components into automated workstations, in order to avoid
cross contamination.
14. Waste produced during the use of the kit has to be
discarded in compliance with national directives and laws
E. MATERIALS REQUIRED BUT NOT PROVIDED
concerning laboratory waste of chemical and biological
1. Calibrated Micropipettes (150ul, 100ul and 50ul) and
substances. In particular, liquid waste generated from the
disposable plastic tips.
washing procedure, from residuals of controls and from samples
2. EIA grade water (double distilled or deionised, charcoal
has to be treated as potentially infective material and inactivated
treated to remove oxidizing chemicals used as
before waste. Suggested procedures of inactivation are
disinfectants).
treatment with a 10% final concentration of household bleach for
3. Timer with 60 minute range or higher.
16-18 hrs or heat inactivation by autoclave at 121°C for 20 min..
4. Absorbent paper tissues.
15. Accidental spills from samples and operations have to be
5. Calibrated ELISA microplate thermostatic incubator (dry or
adsorbed with paper tissues soaked with household bleach and
wet), capable to provide shaking at 1300 rpm+/-150, set at
then with water. Tissues should then be discarded in proper
+37°C.
containers designated for laboratory/hospital waste.
6. Calibrated ELISA microwell reader with 450nm (reading)
16. The Stop Solution is an irritant. In case of spills, wash the
and possibly with 620-630nm (blanking) filters.
surface with plenty of water
7. Calibrated ELISA microplate washer.
17. Other waste materials generated from the use of the kit
8. Vortex or similar mixing tools.
(example: tips used for samples and controls, used microplates)
should be handled as potentially infective and disposed
according to national directives and laws concerning laboratory
wastes.
 Doc.: INS SAG1ULTRA.CE Page 4 of 9 Rev.: 0 Date: 10/2008

G. SPECIMEN: PREPARATION AND WARNINGS 6. Enzyme conjugate:

1. Blood is drawn aseptically by venepuncture and plasma or The working solution is prepared by diluting the 20X
serum is prepared using standard techniques of preparation of concentrated reagent into the Conjugate Diluent, transferred
samples for clinical laboratory analysis. No influence has been from the cartridge into a disposable sterile plastic vial.
observed in the preparation of the sample with citrate, EDTA When the kit is used once for testing in the DP1 ScreenEasy
and heparin. system, open the cartridge containing the Conjugate Diluent,
2. Avoid any addition of preservatives to samples; especially dispense into it 0.8 ml of the 20X conjugate vial, close with the
sodium azide as this chemical would affect the enzymatic plastic cap supplied and mix gently end-over-end for 5 times or
activity of the conjugate, generating false negative results. more.
3. Samples have to be clearly identified with codes or names in Avoid any contamination of the liquid with oxidizing chemicals,
order to avoid misinterpretation of results. When the kit is dust or microbes. If this component has to be transferred, use
used for the screening of blood units, bar code labeling and only plastic sterile disposable containers.
electronic reading is strongly recommended. Important note: The working solution is not stable. Prepare
4. Haemolysed (red) and lipemic (“milky”) samples have to be only the volume necessary for the work of the day. As an
discarded as they could generate false results. Samples example when the kit is used in combination with other
containing residues of fibrin or heavy particles or microbial instruments or manually, dilute 0.1 ml 20X Conjugate with 1.9 ml
filaments and bodies should be discarded as well as they could Conjugate Diluent into a disposable plastic vial and mix carefully
give rise to false positive results. Specimens with an altered before use.
pathway of coagulation, presenting particles after blood
collection and preparation of serum/plasma as those coming 7. Chromogen/Substrate:
from hemodialized patients, could give origin to false positive Ready to use. Mix well by end-over-end mixing.
results. Avoid contamination of the liquid with oxidizing chemicals, air-
5. Sera and plasma can be stored at +2°..8°C for u p to five days driven dust or microbes. Do not expose to strong light, oxidizing
after collection. For longer storage periods, samples can be agents and metallic surfaces.
stored frozen at –20°C for several months. Any fr ozen sample If this component has to be transferred use only plastic, and if
should not be frozen/thawed more than once as this may possible, sterile disposable container.
generate particles that could affect the test result. If some
turbidity is present or presence of microparticles is suspected 8. Sulphuric Acid:
after thawing, filter the sample on a disposable 0.2-0.8u filter to Ready to use. Mix well by end-over-end mixing.
clean it up for testing or use the two-steps alternative method. Attention: Irritant (Xi R36/38; S2/26/30)
Legenda: R 36/38 = Irritating to eyes and skin.
S 2/26/30 = In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice.
H. PREPARATION OF COMPONENTS AND WARNINGS
A study conducted on an opened kit has not pointed out any
relevant loss of activity up to 6 re-uses of the device and up to 6
I. INSTRUMENTS AND TOOLS USED IN COMBINATION
months.
WITH THE KIT
1. Micropipettes have to be calibrated to deliver the correct
1. Microplates:
volume required by the assay and must be submitted to
Allow the microplate to reach room temperature (about 1 hr)
regular decontamination (70% ethanol, 10% solution of
before opening the container. Check that the desiccant has
bleach, hospital grade disinfectants) of those parts that
not turned green, indicating a defect in conservation.
could accidentally come in contact with the sample or the
In this case, call Dia.Pro’s customer service.
components of the kit. They should also be regularly
Unused strips have to be placed back inside the aluminum
maintained in order to show a precision of 1% and a
pouch, with the desiccant supplied, firmly zipped and stored at
trueness of +2%.
+2°..8°C. After first opening, remaining strips are stable until the
2. The ELISA incubator has to be set at +37°C (tolerance of
humidity indicator inside the desiccant bag turns from yellow to
+1°C) and regularly checked to ensure the correct
green.
temperature is maintained. Both dry incubators and water
baths are suitable for the incubations, provided that the
2. Negative Control:
instrument is validated for the incubation of ELISA tests.
Ready to use. Mix well on vortex before use.
3. In case of shaking during incubations, the instrument has to
ensure 350 rpm +150. Amplitude of shaking is very
3. Positive Control:
important as a wrong one could give origin to splashes and
Ready to use. Mix well on vortex before use. The positive
therefore to some false positive result.
control does not contain any infective HBV as it is composed of
4. The ELISA washer is extremely important to the overall
recombinant synthetic HBsAg.
performances of the assay. The washer must be carefully
validated and correctly optimized using the kit
4. Calibrator:
controls/calibrator and reference panels, before using the kit
Add the volume of ELISA grade water, reported on the label, to
for routine laboratory tests. Usually 4-5 washing cycles
the lyophilized powder; let fully dissolve and then gently mix on
(aspiration + dispensation of 350ul/well of washing solution
vortex. The solution is not stable. Store the Calibrator frozen in
= 1 cycle) are sufficient to ensure that the assay performs
aliquots at –20°C.
as expected. A soaking time of 20-30 seconds between
cycles is suggested. In order to set correctly their number, it
5. Wash buffer concentrate:
is recommended to run an assay with the kit
The 20x concentrated solution has to be diluted with EIA grade
controls/calibrator and well-characterized negative and
water up to 1200 ml and mixed gently end-over-end before use.
positive reference samples, and check to match the values
As some salt crystals may be present into the vial, take care to
reported below in the section “Internal Quality Control”.
dissolve all the content when preparing the solution.
Regular calibration of the volumes delivered and
In the preparation avoid foaming as the presence of bubbles
maintenance (decontamination and cleaning of needles) of
could give origin to a bad washing efficiency.
the washer has to be carried out according to the
Note: Once diluted, the wash solution is stable for 1 week at
instructions of the manufacturer.
+2..8° C.
5. Incubation times have a tolerance of +5%.
 Doc.: INS SAG1ULTRA.CE Page 5 of 9 Rev.: 0 Date: 10/2008

6. The microplate reader has to be equipped with a reading 10. Check that the micropipettes are set to the required volume.
filter of 450nm and ideally with a second filter (620-630nm) 11. Check that all the other equipment is available and ready
for blanking purposes. Its standard performances should be to use.
(a) bandwidth < 10 nm; (b) absorbance range from 0 to > 12. In case of problems, do not proceed further with the test and
2.0; (c) linearity to > 2.0; repeatability > 1%. Blanking is advise the supervisor.
carried out on the well identified in the section “Assay
Procedure”. The optical system of the reader has to be
calibrated regularly to ensure that the correct optical density
is measured. It should be regularly maintained according to M. ASSAY PROCEDURE
the manufacturer ‘s instructions. The assay has to be carried out according to what reported
7. When using ELISA automated workstations, all critical below, taking care to maintain the same incubation time for all
steps (dispensation, incubation, washing, reading, shaking, the samples in testing.
data handling, etc.) have to be carefully set, calibrated,
controlled and regularly serviced in order to match the Automated assay:
values reported in the sections “Internal Quality Control”. In case the test is carried out automatically with an ELISA
The assay protocol has to be installed in the operating system, we suggest to make the instrument dispense first 150 ul
system of the unit and validated by checking full matching controls & calibrator, then all the samples and finally 100 ul
the declared performances of the kit. In addition, the liquid diluted Enzyme Conjugate.
handling part of the station (dispensation and washing) has For the pre-washing step (point 1 of the assay procedure) and
to be validated and correctly set paying particular attention all the next operations follow the operative instructions reported
to avoid carry over by the needles used for dispensing below for the Manual Assay.
samples and for washing. The carry over effect must be It is strongly recommended to check that the time lap between
studied and controlled to minimize the possibility of the dispensation of the first and the last sample will be
contamination of adjacent wells due to strongly reactive calculated by the instrument and taken into consideration by
samples, leading to false positive results. The use of ELISA delaying the first washing operation accordingly.
automated work stations is recommended for blood In case the instrument DP1 ScreenEasy is used, follow the
screening and when the number of samples to be tested indications that the computer provides automatically to the user
exceed 20-30 units per run. of DiaPro’s kits when the proper assay protocol is launched.
8. When using the DP1 ScreenEasy workstation, follow
carefully the instructions provided by the instruments itself, Manual Assay:
guiding the operator in all the operative steps. 1. Place the required number of strips in the plastic holder and
9. When using automatic devices, in case the vial holder of the wash them once to hydrate wells. Carefully identify the
instrument does not fit with the vials supplied in the kit, wells for controls, calibrator and samples.
transfer the solution into appropriate containers and label
them with the same label peeled out from the original vial. Important note: Pre washing is fundamental to obtain reliable
This operation is important in order to avoid mismatching and specific results both in the manual and in the automatic
contents of vials, when transferring them. When the test is procedures. Do not omit it !
over, return the secondary labeled containers to 2..8°C,
firmly capped. 2. Leave the A1 well empty for blanking purposes.
10. Dia.Pro’s customer service offers support to the user in 3. Pipette 150µl of the Negative Control in triplicate, 150ul of
the setting and checking of instruments used in combination the Calibrator in duplicate and then 150ul of the Positive
with the kit, in order to assure full compliance with the Control in single followed by 150ul of each of the samples.
essential requirements of the assay. Support is also 4. Check for the presence of samples in wells by naked eye
provided for the installation of new instruments to be used in (there is a marked color difference between empty and full
combination with the kit. wells) or by reading at 450/620nm. (samples show OD
values higher than 0.100).
5. Dispense 100ul diluted Enzymatic Conjugate in all wells,
L. PRE ASSAY CONTROLS AND OPERATIONS except for A1, used for blanking operations.
1. Check the expiration date of the kit printed on the external
label of the kit box. Do not use if expired. Important note: Be careful not to touch the inner surface of the
2. Check that the liquid components are not contaminated by well with the pipette tip when the conjugate is dispensed.
naked-eye visible particles or aggregates. Check that the Contamination might occur.
Chromogen/Substrate is colorless or pale blue. Check that
no breakage occurred in transportation and no spillage of 6. Following addition of the conjugate, check that the color of
liquid is present inside the box. Check that the aluminum the samples have changed from yellowish to red and then
pouch, containing the microplate, is not punctured or incubate the microplate for 120 min at +37°C .
damaged.
3. Dilute all the content of the 20x concentrated Wash Solution Important notes:
as described above. a. Strips have to be sealed with the adhesive sealing foil, only
4. Dilute the 20X concentrated Enzyme Conjugate with its when the test is performed manually. Do not cover strips
Diluent as reported. when using ELISA automatic instruments.
5. Dissolve the Calibrator as described above. b. If the procedure is carried out on shaking, be sure to deliver
6. Allow all the other components to reach room temperature the rpm reported for in Section I.3 as otherwise intra-well
(about 1 hr) and then mix as described. contamination could occur.
7. Set the ELISA incubator at +37°C and prepare the ELISA
washer by priming with the diluted washing solution, 7. When the first incubation is over, wash the microwells as
according to the manufacturers instructions. Set the right previously described (section I.4)
number of washing cycles as found in the validation of the 8. Pipette 200 µl Chromogen/Substrate into all the wells, A1
instrument for its use with the kit. included.
8. Check that the ELISA reader has been turned on at least 20
minutes before reading. Important note: Do not expose to strong direct light as a high
9. If using an automated workstation, turn it on, check settings background might be generated.
and be sure to use the right assay protocol.
 Doc.: INS SAG1ULTRA.CE Page 6 of 9 Rev.: 0 Date: 10/2008

9. Incubate the microplate protected from light at 18-24°C for An example of dispensation scheme is reported in the following
30 min. Wells dispensed with the positive control, the section:
calibrator and positive samples will turn from clear to blue.
10. Pipette 100 µl Sulphuric Acid into all the wells to stop the
enzymatic reaction, using the same pipetting sequence as in Microplate
step 8. Addition of the acid solution will turn the positive
control, the calibrator and positive samples from blue to 1 2 3 4 5 6 7 8 9 10 11 12
yellow. A BLK S2
11. Measure the color intensity of the solution in each well, as B NC S3
described in section I.6 using a 450nm filter (reading) and if C NC S4
possible a 620-630nm filter (background subtraction), D NC S5
blanking the instrument on A1. E CAL S6
F CAL S7
G PC S8
Important general notes:
H S1 S9
Legenda: BLK = Blank NC = Negative Control
1. If the second filter is not available, ensure that no CAL = Calibrator PC = Positive Control S = Sample
fingerprints or dust are present on the external bottom of the
microwell before reading at 450nm. They could generate
false positive results on reading.

2. Reading should ideally be performed immediately after the

addition of the acid solution but definitely no longer than 20 O. INTERNAL QUALITY CONTROL
minutes afterwards. Some self-oxidation of the chromogen A check is performed on the controls/calibrator any time the kit
can occur leading to a higher background. is used in order to verify whether the expected OD450nm or
S/Co values have been matched in the analysis.
3. When samples to be tested are not surely clean or have
been stored frozen, the assay procedure reported below is Ensure that the following results are met:
recommended as long as it is far less sensitive to
interferences due to hemolysis, hyperlipaemia, bacterial Parameter Requirements
contamination and fibrin microparticles. The assay is Blank well < 0.100 OD450nm value
carried out in two-steps at +37°C on shaking at 350 rpm Negative Control (NC) < 0.050 mean OD450nm value after
+150 as follows: blanking
• dispense 100 ul of controls, calibrator and samples Calibrator 0.5 IU/ml S/Co > 2
• incubate 60 min at +37°C on shaking Positive Control > 1.000 OD450nm value
• wash according to instructions (section I.4)
• dispense 100 ul diluted enzyme tracer
• incubate 30 min at +37°C on shaking If the results of the test match the requirements stated above,
• wash proceed to the next section.
• dispense 100 ul TMB&H2O2 mix If they do not, do not proceed any further and perform the
• incubate 30 min at r.t. on shaking following checks:
• stop and read
In this procedure the pre-wash can be omitted.
This method shows performances similar to the standard Problem Check
one and therefore can be used in alternative. Blank well 1. that the Chromogen/Substrate solution
> 0.100 has not become contaminated during the
OD450nm assay
Negative 1. that the washing procedure and the
Control (NC) washer settings are as validated in the
N. ASSAY SCHEME > 0.050 pre qualification study;
OD450nm after 2. that the proper washing solution has
Operations Procedure blanking been used and the washer has been
primed with it before use;
Pre-Washing step n° 1 cycle 3. that no mistake has been done in the
Controls&Calibrator&samples 150 ul assay procedure (dispensation of positive
control instead of the negative one);
Diluted Enzyme Conjugate 100 ul 4. that no contamination of the negative
st
1 incubation 120 min control or of the wells where the control
was dispensed has occurred due to spills
Temperature +37°C of positive samples or of the enzyme
Washing steps n° 4-5 conjugate;
Chromogen/Substrate 200ul 5. that micropipettes have not become
nd contaminated with positive samples or
2 incubation 30 min with the enzyme conjugate
Temperature room 6. that the washer needles are not
blocked or partially obstructed.
Sulphuric Acid 100 ul
Reading OD 450nm
 Doc.: INS SAG1ULTRA.CE Page 7 of 9 Rev.: 0 Date: 10/2008

Calibrator 1. that the procedure has been correctly 2. Any positive result must be confirmed first by repeating the
S/Co < 2 performed; test on the sample, after having filtered it on 0.2-0.8 u filter
2. that no mistake has occurred during its to remove any microparticles interference. Then, if still
distribution (ex.: dispensation of negative positive, the sample has to be submitted to a confirmation
control instead of calibrator) test before a diagnosis of viral hepatitis is released.
3. that the washing procedure and the 3. When test results are transmitted from the laboratory to
washer settings are as validated in the another department, attention must be paid to avoid
pre qualification study; erroneous data transfer.
4. that no external contamination of the 4. Diagnosis of viral hepatitis infection has to be taken and
calibrator has occurred. released to the patient by a suitably qualified medical
Positive Control 1. that the procedure has been correctly doctor.
< 1.000 performed;
OD450nm 2. that no mistake has occurred during An example of calculation is reported below.
the distribution of the control
(dispensation of negative control instead The following data must not be used instead or real figures
of positive control. In this case, the obtained by the user.
negative control will have an OD450nm
value > 0.050). Negative Control: 0.012 – 0.008 – 0.010 OD450nm
3. that the washing procedure and the Mean Value: 0.010 OD450nm
washer settings are as validated in the Lower than 0.050 – Accepted
pre qualification study; Positive Control: 2.489 OD450nm
4. that no external contamination of the Higher than 1.000 – Accepted
positive control has occurred. Cut-Off = 0.010+0.050 = 0.060
Calibrator: 0.350 - 0.370 OD450nm
Mean value: 0.360 OD450nm S/Co = 6.0
If any of the above problems have occurred, report the problem
S/Co higher than 2.0 – Accepted
to the supervisor for further actions.
Sample 1: 0.028 OD450nm
Sample 2: 1.690 OD450nm
Sample 1 S/Co < 0.9 = negative
Sample 2 S/Co > 1.1 = positive
P. CALCULATION OF THE CUT-OFF
The test results are calculated by means of a cut-off value
determined on the mean OD450nm value of the negative control
(NC) with the following formula: R. PERFORMANCE CHARACTERISTICS
Evaluation of Performances has been conducted in accordance
to what reported in the Common Technical Specifications or
NC + 0.050 = Cut-Off (Co) CTS (art. 5, Chapter 3 of IVD Directive 98/79/EC). Version
ULTRA proved to be at least equivalent to the original design in
The value found for the test is used for the interpretation of a study conducted for the validation of the new version.
results as described in the next paragraph.
1. Analytical Sensitivity
Important note: When the calculation of results is performed by The limit of detection of the assay has been calculated on the
the operating system of an ELISA automated work station, nd
2 WHO international standard, NIBSC code 00/588.
ensure that the proper formulation is used to calculate the cut- In the following table, results are given for three lots (P1, P2 and
off value and generate the correct interpretation of results. P3) of the version ULTRA in comparison with the reference
device (Ref.):

WHO Lot # P1 Lot # P2 Lot # P3 Ref.

Q. INTERPRETATION OF RESULTS IU/ml S/Co S/Co S/Co S/Co
Test results are interpreted as a ratio of the sample OD450nm 0.4 4.6 4.8 4.6 4.6
(S) and the Cut-Off value (Co), mathematically S/Co, according 0.2 2.3 2.4 2.4 2.4
to the following table:
0.1 1.4 1.4 1.5 1.2
0.05 0.8 0.8 1.0 0.7
S/Co Interpretation 0.025 0.6 0.6 0.6 0.4
< 0.9 Negative FCS (NC) 0.3 0.2 0.3 0.1
0.9 – 1.1 Equivocal
> 1.1 Positive The assay shows an Analytical Sensitivity better than 0.1 WHO
IU/ml of HBsAg.
A negative result indicates that the patient is not infected by
In addition two panels of sensitivity supplied by EFS, France,
HBV and that the blood unit may be transfused.
and by SFTS, France, were tested and gave in the best
Any patient showing an equivocal result should be retested on a
conditions the following results:
second sample taken 1-2 weeks after the initial sample; the
blood unit should not be transfused.
Panel EFS Ag HBs HB1-HB6 lot n° 04
A positive result is indicative of HBV infection and therefore the
patient should be treated accordingly or the blood unit should be
discarded. Sample ID Characteristics ng/ml S/Co
HB1 diluent / 0,2
HB2 adw2+ayw3 0.05 0,6
HB3 adw2+ayw3 0.1 1,0
Important notes: HB4 adw2+ayw3 0.2 1,8
1. Interpretation of results should be done under the HB5 adw2+ayw3 0.3 2,4
supervision of the laboratory supervisor to reduce the risk of HB6 adw2+ayw3 0.5 4,2
judgment errors and misinterpretations.
 Doc.: INS SAG1ULTRA.CE Page 8 of 9 Rev.: 0 Date: 10/2008

Sensitivity panel SFTS, France, Ag HBs 2005 Results obtained by examining eight panels supplied by Boston
Biomedica Inc., USA, are reported below for the version ULTRA
Sample ID Characteristics ng/ml S/Co in comparison with the reference device code SAG1.CE.
171 Adw2 + ayw3 2.21 + 0.15 15,4
172 Adw2 + ayw3 1.18 + 0.10 8,7 Panel 1st HBsAg HBsAg Version Ref.
ID sample subtype ng/ml ULTRA device
173 Adw2 + ayw3 1.02 + 0.05 6,1
positive S/Co S/Co
174 Adw2 + ayw3 0.64 + 0.04 4,0 PHM 906 02 ad 0.5 3.7 1.4
175 Adw2 + ayw3 0.49 + 0.03 3,4 PHM 907 (M) 06 ay 1.0 4.4 2.9
176 Adw2 + ayw3 0.39 + 0.02 2,6 PHM 909 04 ad 0.3 1.2 0.8
177 Adw2 + ayw3 0.25 + 0.02 2,0 PHM 914 04 ad 0.5 1.1 1.1
178 Adw2 + ayw3 0.11 + 0.02 1,3 PHM 918 02 ad 0.1 1.8 0.5
179 Adw2 + ayw3 0.06 + 0.01 0,9 PHM 923 03 ay < 0.2 2.2 1.2
PHM 925 03 Ind. n.d. 1.4 0.9
180 Adw2 + ayw3 0.03 + 0.01 0,8
PHM 934 01 ad n.d. 1.0 0.8
181 Adw2 0.5 – 1.0 4,7
182 Adw4 0.5 – 1.0 3,6
183 Adr 0.5 – 1.0 4,5 3. Diagnostic Specificity:
184 Ayw1 0.5 – 1.0 5,1 The diagnostic specificity was determined on more than 5000
185 Ayw2 0.5 – 1.0 6,4 negative samples from blood donors (two blood centers),
186 Ayw3 0.5 – 1.0 7,3 classified negative with a CE marked device in use at the
187 Ayw3 0.5 – 1.0 5,8 laboratory of collection and first testing.
188 Ayw4 0.5 – 1.0 6,9 Both plasma, derived with different standard techniques of
189 Ayr 0.5 – 1.0 6,1 preparation (citrate, EDTA and heparin), and sera have been
190 diluent / 0,6 used to determine the specificity.
No false reactivity due to the method of specimen preparation
has been observed.
The panel # 808, supplied by Boston Biomedical Inc., USA, was Frozen specimens have also been tested to check whether
also tested to define the limit of sensitivity. samples freezing interferes with the performance of the test.
Results in the best conditions are as follows : No interference was observed on clean and particle free
samples.
Samples derived from patients with different viral (HCV, HAV)
BBI panel PHA 808 and non viral pathologies of the liver that may interfere with the
test were examined. No cross reaction were observed.
Sample ID Characteristics ng/ml S/Co The study provided a value of Diagnostic Specificity > 99.5% as
01 ad 2,49 10,2 required by the directive 98/79/EC for HBsAg testing .
02 ad 1,17 4,8
03 ad 1,02 4,3 4. Precision:
04 ad 0,96 3,8 It has been calculated for the version ULTRA on two samples
05 ad 0,69 2,9 examined in 16 replicates in 3 different runs for three lots.
06 ad 0,50 2,2 Results are reported in the following tables:
07 ad 0,41 1,5
08 ad 0,37 1,3
09 ad 0,30 1,2 Average values Negative Calibrator
10 ad 0,23 1,0 Total n = 144 Sample 0.5 IU/ml
11 ay 2,51 11,2 OD450nm 0.026 0.332
12 ay 1,26 5,9 Std.Deviation 0.004 0.027
13 ay 0,97 4,1 CV % 16% 8%
14 ay 0,77 3,7
15 ay 0,63 2,0
16 ay 0,48 2,4 The variability shown in the tables did not result in sample
17 ay 0,42 2,0 misclassification.
18 ay 0,33 1,8
19 ay 0,23 1,6
20 ay 0,13 1,1
21 negative / 0,6
S. LIMITATIONS
Repeatable false positive results were assessed on freshly
collected specimens in less than 0.1% of the normal population,
2. Diagnostic Sensitivity: mostly due to high titers Heterophilic Anti Mouse Antibodies
The diagnostic sensitivity was tested according to what required (HAMA).
by Common Technical Specifications (CTS) of the directive Interferences in fresh samples were also observed when they
98/79/EC on IVD for HBsAg testing. were not particles-free or were badly collected (see chapter G).
Positive samples, including HBsAg subtypes and a panel of “s” Old or frozen samples, presenting fibrin clots, crioglobulins,
mutants from most frequent mutations, were collected from lipid-containing micelles or microparticles after storage or
different HBV pathologies (acute, a-symptomatic and chronic thawing, can generate false positive results.
hepatitis B) or produced synthetically, and were detected
positive in the assay.
All the HBsAg known subtypes, “ay” and “ad”, and isoforms “w”
and “r”, supplied by CNTS, France, were tested in the assay and
determined positive by the kit as expected.
An overall value of 100% has been found in a study conducted
on a total number of more than 400 samples positive with the
original reference IVD code SAG1.CE, CE marked.
A total of 30 sero-conversions were studied, most of them
produced by Boston Biomedica Inc., USA.
 Doc.: INS SAG1ULTRA.CE Page 9 of 9 Rev.: 0 Date: 10/2008

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6. Reesink H.W.. et al.. Comparison of six 3 generation tests
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